Biology subjects

Mohammed, J. N.

Publications and source records attributed to Mohammed, J. N..

3 recordsLinked to original sources

bioRxiv2024

A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

Mitochondria maintain a biochemical environment that cooperates with BH3-only proteins (e.g., BIM) to potentiate BAX activation, the key event to initiate physiological and pharmacological forms of apoptosis. The sphingosine-1-phosphate metabolite 2-trans-hexadecenal (2t-hexadecenal) is one such component described to support BAX activation, but molecular mechanisms remain largely unknown. Here, we utilize complementary biochemical and biophysical techniques to reveal that 2t-hexadecenal non-covalently interacts with BAX, and cooperates with BIM to stimulate early-activation steps of monomeric BAX. Integrated structural and computational approaches reveal 2t-hexadecenal binds an undefined region - a hydrophobic cavity formed by core-facing residues of 5, 6, and gated by 8 - we now term the "BAX actuating funnel" (BAF). We define alkenal length and 8 mobility as critical determinants for 2t-hexadecenal synergy with BIM and BAX, and demonstrate that proline 168 allosterically regulates BAF function. Collectively, this work imparts detailed molecular insights advancing our fundamental knowledge of BAX regulation and identifies a regulatory region with implications for biological and therapeutic opportunities.

cell biology↗

bioRxiv2023

An optimized high-yield protocol for expression and purification of monomeric full-length BAX protein

Diverse developmental signals and pro-death stresses converge on regulation of the mitochondrial pathway of apoptosis. BAX, a pro-apoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial member to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX and identify small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally-competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive high-yield protocol for expressing, purifying, and storing functional recombinant BAX protein. We utilize an intein-tagged BAX construct and employ a two-step chromatography strategy to capture and purify BAX, and provide example standard assays to observe BAX activation. We also highlight best practices for handling and storing BAX to effectively preserve its quality, shelf-life, and function.

cell biology↗

bioRxiv2019

Real-time integration of cell death and proliferation kinetics at the single-cell and population-level using high-throughput live-cell imaging.

Quantifying cytostatic and cytotoxic outcomes are integral components of characterizing perturbagens used as research tools and/or in drug discovery pipelines. Furthermore, data-rich acquisition coupled with robust methods for analysis is required to properly assess the function and impact of these perturbagens. Here, we present a detailed and versatile method for Single-cell and Population-level Analyses using Real-time Kinetic Labeling (SPARKL). SPARKL integrates high-content live-cell imaging with automated detection and analysis of fluorescent reporters of cell death. We outline several examples of zero-handling, non-disruptive protocols for detailing cell death mechanisms and proliferation profiles. Additionally, we suggest several methods for mathematically analyzing these data to best utilize the collected kinetic data. Compared to traditional methods of detection and analysis, SPARKL is more sensitive, accurate, and high-throughput while substantially eliminating sample processing and providing richer data.

cell biology↗